Natural killer (NK) cells represent an important component of the immune system. They are a group of white blood cells (lymphocytes) that can directly kill virus-infected cells and tumor cells. NK cells are essential for resistance to many pathogens, such as viruses, bacteria, and parasites. Unlike B and T cells, NK cells do not express antigen-specific receptors, which raises the interesting question of how their specificity toward target cells is determined. NK cells exert their function in two ways: by producing cytokines, such as interferon-gamma, and by killing infected cells. Despite the importance of NK cells in the innate immune response to many types of pathogens, it is still unclear what receptors and what signal transduction pathways control their activation. The major goal of this project is to define receptor-ligand interactions that regulate cytotoxicity (lysis of target cells) and cytokine production by NK cells, and to determine the precise contribution of individual receptors to signal transduction in NK cells. Natural killer (NK) cells provide innate control of infected and neoplastic cells. Multiple receptors have been implicated in natural cytotoxicity, but their individual contributions to this process remain unclear. We have studied the activation of resting human NK cells by Drosophila cells expressing ligands for the receptors NKG2D, DNAM-1, 2B4, CD2, and the integrin LFA-1. Each receptor was capable of inducing inside-out signals for LFA-1, which promoted adhesion. In contrast, none of these receptors alone was able to induce degranulation. Rather, release of cytolytic granules was induced by synergistic activation through co-engagement of receptors, as shown for NKG2D and 2B4. Whereas engagement of NKG2D and 2B4 did not induce strong target cell lysis, collective engagement of LFA-1, NKG2D, and 2B4 defined a minimal requirement for induction of natural cytotoxicity. Remarkably, inside-out signaling to LFA-1 induced by each receptor, including LFA-1 itself, was blocked by co-engagement of inhibitory receptor CD94/NKG2A. Inside-out signals induced by the NKG2D and 2B4 combination could overcome the inhibition by CD94/NKG2A. These results detail how activating receptors for natural cytotoxicity co-operate for immunosurveillance and reveal that signals as proximal as inside-out signaling for adhesion are sensitive to inhibition by CD94/NKG2A. By employing Drosophila cells that express ligands of NK cell activation receptors, in the absence of anti-receptor antibodies and exogenous cytokines, we have defined co-engagement of receptors NKG2D, 2B4, and LFA-1 as a minimal requirement for natural cytotoxicity mediated by normal, freshly isolated, resting NK cells. Therefore, our results revealed that target cell recognition by NK cells is a highly dynamic process controlled by the integration of signals from multiple receptors, many of which promote adhesion, some of which synergize to induce degranulation, and all of which are sensitive to inhibitory signaling by receptor CD94/NKG2A. Natural killer (NK)cell recognition of infected or neoplastic cells can induce cytotoxicity and cytokine secretion. So far, it has been difficult to assess the relative contribution of multiple NK-cell activation receptors to cytokine and chemokine production upon target cell recognition. Using Drosophila cells expressing ligands for the NK-cell receptors LFA-1, NKG2D, DNAM-1, 2B4, and CD16, we studied the minimal requirements for secretion by freshly isolated, human NK cells. Target cell stimulation induced secretion of predominately proinflammatory cytokines and chemokines. Release of chemokines MIP-1alpha&#1113090;, MIP-1beta, and RANTES was induced within 1 hour of stimulation, whereas release of TNF-alpha and IFN-gamma occurred later. Engagement of CD16, 2B4, or NKG2D sufficed for chemokine release, whereas induction of TNF-alpha&#1113090;and IFN-gamma required engagement of additional receptors. Remarkably, our results revealed that, upon target cell recognition, CD56dim NK cells were more prominent cytokine and chemokine producers than CD56bright NK cells. The present data demonstrate how specific target cell ligands dictate qualitative and temporal aspects of NK-cell cytokine and chemokine responses. Conceptually, the results point to CD56dim NK cells as an important source of cytokines and chemokines upon recognition of aberrant cells, producing graded responses depending on the multiplicity of activating receptors engaged. This study has provided detailed insight into regulation of NK-cell cytokine secretion upon target cell recognition. Engagement of individual activating receptors on resting NK cells suffices for chemokine secretion, to alert and recruit other immune cells. More complex interactions, upon which multiple activating NK- cell receptors are engaged, can induce production of TNF-alpha and IFN-gamma, to promote cellular resistance to infections and shape adaptive immune responses. These chemokines and cytokines are readily produced by the CD56dim NK-cell subset. Chemokine and cytokine production by CD56dim NK cells may thus be an important component of immunosurveillance. Both T and NK cells kill target cells through polarized exocytosis of lytic granules, which contain death-inducing proteins such as perforin, granzymes, and Fas ligand. Granule exocytosis involves polarization towards the immunological synapse, docking at the plasma membrane, and fusion with the plasma membrane. Little is known about the movement of lytic granules at steady-state in unstimulated cytotoxic lymphocytes. Even in the absence of NK cell stimulation by target cells or by cell surface receptors, some of the lytic granules are close to the plasma membrane, as visualized by total internal reflection fluorescence (TIRF) microscopy. Lytic granules that are close to the plasma membrane may represent a functional pool available for release of cytolytic effectors, as degranulation by NK cells has been observed in the absence of granule polarization. Rab27a, a Ras-like GTPase protein, is defective in patients with Griscelli Syndrome type 2, which is an autosomal recessive, rare immune disorder associated with hypopigmentation, impaired cytotoxicity, and with poor docking of lytic granules at the plasma membrane. We examined how Rab27a regulates LG movement both at the plasma membrane and in the cytosol of human and of mouse NK cells in the absence of activation signals. Protocols were developed to automate image analysis and to track the movement of thousands of vesicular compartments in live cells. Algorithms were used to discriminate among different types of movement (e.g. random, caged, and directed). We applied these tools to investigate the steady-state distribution and movement of lytic granules in live NK cells by high-speed 3-dimensional spinning disc confocal and 2-dimensional total internal reflection fluorescence microscopy. Mouse NK cells and a human NK cell line deficient in the small GTPase Rab27a were also examined. The unbiased analysis of large datasets led to the following observations and conclusions. A majority of lytic granules in the cytosol and at the plasma membrane of unstimulated NK cells are mobile. Inhibitors indicated that movement in the cytosol required microtubules but not actin, whereas movement under the plasma membrane required both. Rab27a deficiency resulted in fewer lytic granules at the plasma membrane and in a reduced fraction of mobile lytic granules. In contrast, loss of Rab27a increased the fraction of mobile lytic granules and the extent of their movement in the cytosol. Therefore, in addition to its documented role in lytic granules delivery to the plasma membrane, Rab27a may serve as tether to constrain lytic granules movement in the cytosol.